The invention relates to derivatives of poly(ethylene glycol) and related polymers and methods for their synthesis. More particularly, the invention relates to high molecular weight derivatives and methods of producing high molecular weight derivatives.
Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated PEG, also known as poly(ethylene oxide), abbreviated PEO, to molecules and surfaces is of considerable utility in biotechnology and medicine. In its most common form, PEG is a linear polymer terminated at each end with hydroxyl groups:
HOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH
The above polymer, xcex1, xcfx89-dihydroxypoly(ethylene glycol), can be represented in brief form as HO-PEG-OH where it is understood that the -PEG- symbol represents the following structural unit:
xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94
where n typically ranges from about 3 to about 4000.
PEG is commonly used as methoxy-PEG-OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification. The structure of mPEG is given below.
CH3Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH
The copolymers of ethylene oxide and propylene oxide are closely related to PEG in their chemistry, and they can be substituted for PEG in many of its applications.
HOxe2x80x94CH2CHRO(CH2CHRO)nCH2CH2xe2x80x94OH
where Rxe2x95x90H or alkyl, such as CH3.
PEG is also commonly used in multi-arm forms in which linear PEGs are attached to a central core:
R(xe2x80x94O-PEG-OH)n
where R is a core derived from, for example, pentaerythritol or glycerol oligomers. PEGs can also be prepared with degradable linkages in the backbone.
PEG is a polymer having the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PEG is to covalently attach the polymer to insoluble molecules to make the resulting PEG-molecule xe2x80x9cconjugatexe2x80x9d soluble. For example, it has been shown that the water-insoluble drug paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336 (1995).
To couple PEG to a molecule, such as a protein, it is often necessary to xe2x80x9cactivatexe2x80x9d the PEG to prepare a derivative of the PEG having a functional group at the terminus. The functional group can react with certain moieties on the protein, such as an amino group, thus forming a PEG-protein conjugate. Many activated derivatives of PEG have been described. An example of such an activated derivative is the succinimidyl succinate xe2x80x9cactive esterxe2x80x9d:
CH3O-PEG-O2Cxe2x80x94CH2CH2xe2x80x94CO2xe2x80x94NS
where NS=
Hereinafter, the succinimidyl active ester moiety will be represented as xe2x80x94CO2xe2x80x94NS. Such activated PEGs can also be prepared from the above described multi-arm forms or from branch forms such as:
(PEG-O-COxe2x80x94NH)2LYS-NS
as described in Harris, et al., U.S. Pat. No. 5,932,462, which is incorporated by reference herein in its entirety. Functional groups can be attached to the terminus of PEG by direct conversion of the starting hydroxyl to other forms or by attachment of organic spacer groups to the hydroxyl group. For example, the succinate PEG above is prepared by attachment of succinic anhydride to PEG. Similarly one can react glutaric anhydride to prepare PEG glutarate, PEG-O2Cxe2x80x94CH2CH2CH2xe2x80x94CO2H. Even larger aliphatic spacers can be added. As described in Okamoto, et al., Eur. Polym. J., 19, 341-346 (1983), PEG can be converted to a PEG amine by reacting PEG-OH with ONCxe2x80x94(CH2)6xe2x80x94NCO and then converting the remaining isocyanate to amine product PEG-O2CNHxe2x80x94(CH2)6xe2x80x94NH2.
As applications of PEG chemistry have become more sophisticated, there has been an increasing need for high molecular weight, high purity PEG derivatives. The synthesis of these compounds is complicated by the difficulty in removing polymeric impurities that accumulate during multi-step preparations. Small molecule impurities are normally easily removed by simple procedures such as precipitation. However, high molecular weight polymeric side-products are generally quite difficult to remove and require utilization of time-consuming and expensive chromatographic techniques. There remains a need in the art for improved methods of preparing high molecular weight PEG derivatives.
The invention includes high molecular weight activated polymer derivatives and methods for making them. A small, difunctional oligo(ethylene glycol) (xe2x80x9cOEGxe2x80x9d) derivative or similar oligomer or small polymer is covalently linked to a large poly(ethylene glycol) polymer (xe2x80x9cPEGxe2x80x9d) derivative or similar polymer. In this way, most of the chemical transformations can be conducted on the oligomeric or small polymeric compound. Large polymeric impurities are more difficult to separate from the desired product than are smaller ones, and the products of these reactions involving these polymers typically include unreacted reagents, difunctional components that can result in cross linking, partially reacted components, and other polymeric impurities. The invention avoids these impurities by reducing the number of reactions needed to create the large polymer.
Thus, as shown below, one can make a complicated polymeric compound in one step by reacting a complicated oligomer, for example, Yxe2x80x2xe2x80x94OEG-Y, where Y and Yxe2x80x2 are active moieties, with a simple high molecular weight polymeric compound, for example, mPEG-Z, where Z reacts with one of Y or Yxe2x80x2, to make a new linking group X between the larger PEG and the smaller PEG. In this way, polymeric impurities do not accumulate. Y and Yxe2x80x2 can be the same or different, but should be chosen so that the two moieties are compatible and will not react with each other.
In a typical reaction for producing a high molecular weight monofunctional large molecule, a monomethoxy poly(ethylene glycol) is reacted with a smaller PEG polymer, in which the functional group Z on the monofunctional larger PEG polymer reacts with the functional group Yxe2x80x2 on the heterobifunctional smaller polymer. The high molecular weight product retains the active group Y. The reactants are linked by a group X formed by the reaction of the Z and Yxe2x80x2 moieties. This reaction can be illustrated as follows:
mPEG-Z+Yxe2x80x2-OEG-Yxe2x86x92mPEG-X-OEG-Y
For example, an mPEG-propionic acid of molecular weight 32,000 which is a compound typically made in several steps from mPEG-OH, can be prepared in a single step by reacting an activated MPEG carbonate 30,000 that has been prepared in one step from mPEG-OH, with xcex1-amino-xcfx89-propionic acid of molecular weight 2000. Most of the chemical transformations can be performed on the small, inexpensive, more readily purified PEG 2000.
Monofunctional, homobifunctional, and heterobifunctional large molecules can be prepared by the practice of the invention, although not necessarily all with equivalent results. Reactions that tend to introduce complications in the larger polymeric component of the product molecule may reduce the effectiveness of the method if impurities are increased with multiple reaction steps.
In a somewhat more generalized embodiment, showing a poly(ethylene glycol) polymer with greater specificity, the structure of the products of the invention can be described as follows:
Rxe2x80x94(OCH2CH2)nxe2x80x94Xxe2x80x94(CH2CH2xe2x80x94O)mxe2x80x94Y
The above structure is prepared by reacting Rxe2x80x94(OCH2CH2)nxe2x80x94Z with Yxe2x80x2xe2x80x94(CH2CH2xe2x80x94O)mxe2x80x94Y, where Z is a group reactive with Yxe2x80x2 and neither Y nor R is reactive with Z or Yxe2x80x2. R can be a capping moiety, including various alkyl moieties, typically methoxy as attached to PEG. R can also be a reactive group or a protected reactive group Yxe2x80x3 in which the reactive group can be deprotected and available for reaction at some later desired time. Y and Yxe2x80x2 can be the same if Yxe2x80x2 has been a protected group or a different group that does not participate in the reactions used to create the product molecule. Functional groups include, but are not limited to, aldehyde, maleimide, active ester, thiosulfonate or amine. X is a small, hydrophilic linker such as amide or carbamate; n is from about 200 to 2000; and m is from about 1 to 120. X is desirably a small and hydrophilic moiety that will not adversely impact the chemical and physical properties of the resulting high molecular weight polymer.
Alternatively, a large polymeric segment that is bifunctional or has additional functionality can be linked to smaller molecules at one or more functionalities. The functionality of the product large molecule can originate from the large polymeric segment or the smaller oligomeric or polymeric segment, as desired. The polymer backbone can have groups or linkers subject to hydrolysis or enzymatic degradation built into the backbone if desired for controlled degradability of the product molecule.
The polymer segments above may broadly be thought of as (Poly)a for the larger polymer segment and (Poly)b for the smaller polymer segment where poly can be any of the polymers including poly(ethylene glycol) in its various forms and polymers considered in the art to be of similar characteristics.
Thus, a high molecular weight polymer can be prepared from a lower molecular weight polymer, from which it is much less troublesome to remove impurities, and a higher molecular weight polymer that has not been subjected to a series of reactions or complex purification steps to remove polymeric impurities.
The present invention now will be described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Definitions
The terms xe2x80x9cfunctional group,xe2x80x9d xe2x80x9cactive moiety,xe2x80x9d xe2x80x9cactivating group,xe2x80x9d xe2x80x9creactive site,xe2x80x9d xe2x80x9cendgroup,xe2x80x9d xe2x80x9cchemically reactive group,xe2x80x9d and xe2x80x9cchemically reactive moietyxe2x80x9d are used in the art and herein to refer to distinct, definable portions or units of a molecule. The terms are somewhat synonymous in the chemical arts and are used herein to indicate the portions of molecules that perform some function or activity and are reactive with other molecules. The term xe2x80x9cactive,xe2x80x9d when used in conjunction with functional groups, is intended to include those functional groups that react readily with electrophilic or nucleophilic groups on other molecules, in contrast to those groups that require strong catalysts or highly impractical reaction conditions to react (i.e., xe2x80x9cnon-reactivexe2x80x9d or xe2x80x9cinertxe2x80x9d groups). For example, as would be understood in the art, the term xe2x80x9cactive esterxe2x80x9d would include those esters that react readily with nucleophilic groups such as amines. Exemplary active esters include N-hydroxysuccinimidyl esters or 1-benzotriazolyl esters. Typically, an active ester will react with an amine in aqueous medium in a matter of minutes, whereas certain esters, such as methyl or ethyl esters, require a strong catalyst in order to react with a nucleophilic group.
The term xe2x80x9calkylxe2x80x9d refers to hydrocarbon chains typically ranging from about 1 to about 12 carbon atoms in length, and includes straight and branched chains. The hydrocarbon chains may be saturated or unsaturated. The term xe2x80x9csubstituted alkylxe2x80x9d refers to an alkyl group substituted with one or more non-interfering substituents, such as, but not limited to, C3-C6 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; acetylene; cyano; alkoxy, e.g., methoxy, ethoxy, and the like; lower alkanoyloxy, e.g., acetoxy; hydroxy; carboxyl; amino; lower alkylamino, e.g., methylamino; ketone; halo, e.g. chloro or bromo; phenyl; substituted phenyl, and the like.
As used herein, xe2x80x9cnon-peptidicxe2x80x9d refers to a polymer backbone substantially free of peptide linkages. However, the polymer backbone may include a minor number of peptide linkages spaced along the length of the backbone, such as, for example, no more than about 1 peptide linkage per about 50 monomer units.
A xe2x80x9cpolymer conjugatexe2x80x9d refers to a water soluble polymer backbone covalently attached to a biologically active molecule, as defined herein. In the case that a polymer conjugate is reacted with a second polymer so as to form an extended polymer backbone, whether or not the joinder of the polymers is with a peptidic or other linkage, the term xe2x80x9cpolymer conjugatexe2x80x9d refers to the overall length of polymer bound to the biologically active agent.
The term xe2x80x9clinkagexe2x80x9d, xe2x80x9clinkerxe2x80x9d, or xe2x80x9clinking groupxe2x80x9d is used herein to refer to groups or bonds that normally are formed as the result of a chemical reaction and typically are covalent linkages. Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at useful pHs, e.g., under physiological conditions for an extended period of time, preferably indefinitely.
Hydrolytically unstable or degradable linkages means that the linkages are degradable in water or in aqueous solutions, including for example, blood. Enzymatically unstable or degradable linkages means that the linkage can be degraded by one or more enzymes.
The term xe2x80x9cbiologically active moleculexe2x80x9d, xe2x80x9cbiologically active moietyxe2x80x9d, xe2x80x9cbiologically active agentxe2x80x9d, or xe2x80x9cdrugxe2x80x9d when used herein means any substance which can affect any physical or biochemical properties of a biological organism, including but not limited to viruses, bacteria, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include any substance intended for diagnosis, cure mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, dyes, lipids, nucleosides, oligonucleotides, cells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the invention include, but are not limited to, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, and the like.
The terms xe2x80x9clow weight polymerxe2x80x9d and xe2x80x9clow molecular weight polymerxe2x80x9d broadly refer to a linear, branched, multi-arm, or forked polymer backbones comprising a water-soluble and non-peptidic polymer having from 1 to about 120 repeating units. These polymers typically have from 1 to 2 functional groups, typically located at opposite termini on a linear polymer, to about 300, which can be located at the termini of highly branched or multiarmed structures, although a smaller number may be located along the polymer backbone. Suitable polymers include, but are not limited to poly(alkylene glycol), such as poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly((xcex1-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, and copolymers, terpolymers, derivatives and mixtures thereof. It should be understood that oligomeric and even monomeric lengths are to be included in the above. Although the molecular weight of the small polymer or oligomer can vary, it is typically in the range of from about 100 Da to about 10,000 Da, depending, of course, on the molecular weight of the individual repeating units. In the case of PEG, one PEG monomer unit has a molecular weight of about 44 Da and low weight polymers will have a molecular weight of from about 44 Da to about 5280 Da. Molecular weights of 2000, 3200, 3400, and 5,000 are commonly available commercially. Those of ordinary skill in the art will recognize that the foregoing list for substantially water soluble and non-peptidic polymer backbones is by no means exhaustive and is merely illustrative, and that all polymeric materials having the qualities described above are contemplated.
The terms xe2x80x9chigh weight polymerxe2x80x9d and xe2x80x9chigh molecular weight polymerxe2x80x9d broadly refer to a linear, branched, or multi-arm polymer backbone comprising a water-soluble and non-peptidic polymer having more than about 200 repeating units. These polymers typically have from 1 to 2 functional groups, typically located at opposite termini on a linear polymer, to about 300, which can be located along the polymer backbone or at the termini of highly branched or multiarmed structures. Forked structures are also contemplated in which a terminus is branched to provide two functionalities. Suitable polymers include, but are not limited to these same polymers from which the low weight polymer is selected. Although the molecular weight of the polymer can vary, it is typically greater than about 8,000 Da depending, of course, on the molecular weight of the individual monomer units. In the case of PEG, high weight polymers have a molecular weight above about 8,800 Da. Commercially available PEGs include those having a nominal molecular weight of 10,000, 12,000, 15,000, 18,000, and 20,000, 30,000, 40,000 and above. Branched PEGs are readily available at higher molecular weights. Those of ordinary skill in the art will recognize that the foregoing list for substantially water soluble and non-peptidic polymer backbone is by no means exhaustive and is merely illustrative, and that all polymeric materials having the qualities described above are contemplated.
As used herein, xe2x80x9cPEGxe2x80x9d broadly refers to a linear, multi-arm, or branched polymer backbone comprising a water-soluble and non-peptidic polymer having repeat CH2CH2O units. The polymer xcex1, xcfx89-dihydroxypoly(ethylene glycol), can be represented in brief form as HO-PEG-OH where it is understood that the -PEG- symbol represents the following structural unit xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94 where n typically ranges from about 3 to about 2000. The PEG family of polymers generally exhibits the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. The term PEG should be understood to be inclusive and to include poly(ethylene glycol) in any of its linear, branched or multi-arm forms, including alkoxy PEG, bifunctional PEG, forked PEG, branched PEG, pendant PEG, or PEG with degradable linkages therein.
PEG, in any of the forms described herein, is typically clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze or deteriorate (unless specifically designed to do so), and is generally non-toxic. Poly(ethylene glycol) is considered to be biocompatible, which is to say that PEG is capable of coexistence with living tissues or organisms without causing harm. More specifically, PEG is substantially non-immunogenic, which is to say that PEG does not tend to produce an immune response in the body. When attached to a molecule having some desirable function in the body, such as a biologically active agent, the PEG tends to mask the agent and can reduce or eliminate any immune response so that an organism can tolerate the presence of the agent. PEG conjugates tend not to produce a substantial immune response or cause clotting or other undesirable effects.
Random or block copolymers of ethylene oxide and propylene oxide, shown of either the high weight or low weight polymers of the invention wherein -PEG- is replaced with:
xe2x80x83xe2x80x94CH2CHRO(CH2CHRO)cCH2CHRxe2x80x94
and wherein each R is independently H or CH3, and c is as described above for m and n, depending on whether the molecule is a small or large one.
The term xe2x80x9cpolymer derivativexe2x80x9d is sometimes used to describe the high weight polymer product of the invention having a high weight polymer segment and a low weight polymer segment joined by a linkage. The term is merely one of convenience, and is used to differentiate the product polymer from its high weight and low weight polymer components when so indicated.
In one form useful in the invention, the high molecular weight polymer is linear PEG terminated at one end with a hydroxyl group and the other end with a functional group, Z:
HOxe2x80x94CH2CH2O xe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94Z
The above polymer can be represented in brief form as HO-PEG-Z where it is understood that the -PEG- symbol represents the following structural unit:
xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94
where n is greater than 200, and typically ranges from about 200 to 2000 for the high weight polymer. This polymer can also be represented more generically as Rxe2x80x94(Poly)axe2x80x94Z, where (Poly)a stands for the large polymer component of the invention and R is a capping moiety or a suitable reactive group or protected reactive group Yxe2x80x3.
Another type of PEG useful as the high weight polymer of the invention, (Poly)a is methoxy-PEG-Z, or mPEG-Z in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is the functional group, Z. The structure of MPEG is given by:
CH3Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94Z
where n is as described above. The use of an mPEG having a functional group, Z, or having other active sites of the polymer than Z capped by alkyl, aryl, or other non-reactive groups or protective groups prevents the high weight polymer from self-polymerizing and from undesirably combining with other molecules in the reaction mixture.
The functional group, Z, of the high weight polymer is selected such that the Z group readily forms a covalent bond with a corresponding reactive species, Y or Yxe2x80x2, found on the low weight polymer thereby forming a linkage, X, between the high weight polymer and low weight polymer when reacted. Suitable functional groups are discussed below.
As an exemplary form useful in the practice of the invention, the low molecular weight polymer is a linear PEG polymer terminated at one end with a functional group, Y or Yxe2x80x2, capable of readily forming a hydrolytically stable linking group when reacted with the Z component of the high weight polymer, and an additional functional group, Y or Yxe2x80x2, capable of forming a covalent bond with a biologically active agent or of being modified to a form which is capable of forming a covalent bond with a biologically active agent:
Yxe2x80x2xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)mxe2x80x94CH2CH2xe2x80x94Y
The above polymer can be represented in brief form as Yxe2x80x2-PEG-Y where it is understood that the -PEG- symbol represents the following structural unit:
xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)mxe2x80x94CH2CH2xe2x80x94
where m ranges from 1 to about 120, and is typically less than about 60, for the low weight polymer. More generically, the polymer can be represented as Yxe2x80x2-(Poly)b-Y, where (Poly)b stands for the small polymer component of the invention. The functional groups Yxe2x80x2 and Y of the low weight polymer are selected so that they do not readily bond with each other under reaction conditions, thus avoiding self-polymerization of the low weight polymer. Acceptable Yxe2x80x2 and Y groups are specified more fully below. below, are closely related to PEG in their chemistry, and can also be used as the polymer backbone
The polymer backbones may also comprise a branched structure, typically having a central branching core moiety and a plurality of polymer chains, preferably linear polymer chains, linked to the central core. In one embodiment, PEG is used in a branched form prepared, for example, by addition of ethylene oxide to various polyol central core structures, such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. Any polyol providing a plurality of hydroxyl groups available for conjugation to polymer chains may be used in the practice of the invention. The polyol branching core structure can provide from about 3 to 100 available hydroxy groups, and typically provides about 3 to 20, so that the branched polymer structure has from about 3 to 100 polymer chains. The branched poly(ethylene glycol) molecules of this type can be represented in general form as R(-PEG-OH)a in which R is derived from a central core moiety, such as glycerol, glycerol oligomers, or pentaerythritol, and xe2x80x9caxe2x80x9d represents the number of arms, typically about 3 to 20.
For use as the high molecular weight polymer, the hydroxyl groups of the branched PEG normally are converted to functional groups Z prior to combination with the low weight polymer. Branched or multi-armed high weight polymers have the form R(-PEG-Z)a, and are capable of bonding to about 3 to 20 of the low weight polymers of the invention, depending on the degree of branching or the number of arms having active groups. The central core moiety of the branched molecule can be derived from any of a number of amino acids, such as lysine, wherein the central core moiety typically provides two or more sites, e.g., amino groups, for attachment of polymer chains. Multi-armed PEG molecules, such as those described in U.S. Pat. No. 5,932,462, which is incorporated by reference herein in its entirety, can also be used as the polymer backbone.
The polymer backbone of the high molecular weight polymer may alternatively comprise a forked PEG. An example of a forked PEG is represented by PEG-ACHZ2, where A is a linking group and Z is an activated terminal group linked to CH by a chain of atoms of defined length. International Application No. PCT/US99/05333, the contents of which are incorporated by reference herein, discloses various forked PEG structures for use in one embodiment of the invention. The chain of atoms linking the Z functional groups to the branching carbon atom serve as a tethering group and may comprise, for example, alkyl chains, ether chains, ester chains, amide chains and combinations thereof. The Z functional groups can be used in the present invention to react with one of the functional groups, Y or Yxe2x80x2, on the low weight polymer to form a linkage between the low weight and the high weight polymers.
The polymer backbone of either the low or high weight polymer may comprise a pendant PEG molecule having functional groups, Z, Y, or Yxe2x80x2 as the case may be, covalently attached along the length of the PEG backbone rather than at the end of the PEG chain. The pendant reactive groups can be attached to the PEG backbone directly or through a linking moiety, such as alkylene.
In the case of a high weight polymer having more than one functional group, Z, there are many sites along the polymer where the corresponding functional group, Y or Yxe2x80x2, on the low weight polymer may bond. The polymer derivatives produced therefrom are represented for convenience as:
PEG-[Xxe2x80x94(CH2CH2O)mxe2x80x94Y]q
wherein PEG is a linear poly(ethylene glycol), alkoxy-poly(ethylene glycol), a branched poly(ethylene glycol), or a forked poly(ethylene glycol) where the PEG has a degree of polymerization of at least about 200, preferably greater than about 1000, and preferably less than about 2000, X is the linking moiety, m is from 1 to about 120, and preferably less than 60, Y is a functional group for attachment to a biologically active agent, and q is equal to the number of polymer end groups and can be from 1 to about 500. Where q is 2, then the Y""s can be the same or different, but if q is much greater than 3 or 4, the Y""s would normally all be the same group. More generically, in the structure above, PEG can be (Poly)a and the monomer unit CH2CH2O can be represented as (Poly)b to include similar small polymers, copolymers, oligomers, and the like.
In addition to the above-described forms of PEG, any of the above polymers can also be prepared with one or more weak or degradable linkages in the backbone. For example, PEG can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer into fragments of lower molecular weight:
-PEG-CO2-PEG-+H2O xe2x86x92-PEG-CO2H+HO-PEG-
Similarly, a polymer backbone can be covalently attached to a biologically active agent through a weak or degradable linkage moiety. For example, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent generally hydrolyze under physiological conditions to release the agent.
Other hydrolytically degradable linkages, useful as either a degradable linkage within a polymer backbone or as a degradable linkage connecting a polymer backbone to a biologically active agent, include carbonate linkages; imine linkages resulting, for example, from reaction of an amine and an aldehyde (see, e.g., Ouchi et al., Polymer Preprints, 38(1):582-3 (1997), which is incorporated herein by reference.); phosphate ester linkages formed, for example, by reacting an alcohol with a phosphate group; hydrazone linkages which are typically formed by reaction of a hydrazide and an aldehyde; acetal linkages that are typically formed by reaction between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction between a formate and an alcohol; peptide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by, for example, a phosphoramidite group, e.g., at the end of a polymer, and a 5xe2x80x2 hydroxyl group of an oligonucleotide.
Many other polymers are also suitable for the invention. Polymer backbones that are non-peptidic and water-soluble are particularly useful in the invention. Examples of suitable polymers that may be used in place of PEG in the manner specified above include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol and the like, poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(xcex1-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), such as described in U.S. Pat. No. 5,629,384, which is incorporated by reference herein in its entirety, and copolymers, terpolymers, and mixtures thereof.
Those of ordinary skill in the art will recognize that the foregoing list for substantially water soluble polymers is by no means exhaustive and is merely illustrative, and that all polymeric materials having the qualities described above are contemplated.
Components Forming the X Linking Group
As mentioned, the method and resulting functionalized polymer can be represented by:
R-(Poly)a-Z+Yxe2x80x2-(Poly)b-Yxe2x86x92Y-(Poly)b-X-(Poly)a-X-(Poly)bxe2x80x94Y
Z is a functionalized endgroup of the high weight polymer (Poly)a and Y and Yxe2x80x2 are functionalized endgroups of the low weight polymer (Poly)b. R can include a functionalized end group Z if it is desired to produce a homobifunctional polymer as shown, or a protected active group for later coupling to another activated smaller polymer, activated with a different group Yxe2x80x3, if a heterobifunctional large polymer is desired.
Various reactive groups Y, Yxe2x80x2, Yxe2x80x3 and Z include but are not limited to, N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al. Makromol.Chem. 182:1379 (1981), Zaplipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g., Olson et al. in Poly(ethylene glycol) Chemistry and Biological Applications, pp 170-181, Harris and Zaplipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al. Macrolol. Chem. 180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem. Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan, Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of the above references are incorporated herein by reference.
The endgroups Z and the Y, Yxe2x80x2 or Yxe2x80x3 groups reactive with Z are selected such that the endgroup moieties are complementary, meaning that the endgroups readily react with one another under reaction conditions to form a linking group X which is typically hydrolytically stable under physiological conditions. It is the formation of the linking group X which binds the high molecular weight polymer to the low molecular weight polymer, thereby forming an overall high molecular weight functionalized polymer derivative. Y, Yxe2x80x2, and Yxe2x80x3 can be the same or different so long as they are not reactive with each other. Additionally, at least one reactive group Y or Yxe2x80x2 should not react with Z so as to provide functionality for the completed polymer.
The functional groups or chemically reactive groups on the low weight polymer may be selected from the group consisting of hydroxyl, protected hydroxyl, active ester, active carbonate, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, protected amine, hydrazide, protected hydrazide, thiol, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates, thiosulfonate, and tresylate.
Reactive endgroups can be selected from one or a combination of the following: xe2x80x94(CH2)rCO2H, xe2x80x94(CH2)rCO2NS, xe2x80x94(CH2)rCO2Bt, xe2x80x94(CH2)rCH(OR)2, xe2x80x94(CH2)rCHO, xe2x80x94(CH2)2xe2x80x94NH2, xe2x80x94(CH2)rM, xe2x80x94(CH2)rxe2x80x94Sxe2x80x94SO2xe2x80x94R, where r is 1-5, rxe2x80x2 is 0-5, R is aryl or alkyl, NS is N-succinimidyl, Bt is 1-benzotriazolyl, and M is N-maleimidyl.
Typically, the linking moiety X is hydrophilic and short. However, the linking group could be prepared from hydrophobic components so long as, whether hydrophilic or hydrophobic, the linking group does not substantially alter the properties of the polymer. That is, in the case of PEG, the linking group should not alter the properties as set forth above in the definition of PEG.
Reaction Conditions
Suitable solvents providing a medium for the reaction of the Z and Y, Yxe2x80x2, or Yxe2x80x3 endgroups in formation of the X linking group include but are not limited to toluene, tetrahydrofuran, dioxane, acetonitrile, methylene chloride, chloroform, dimethylformamide, dimethylsulfoxide, benzene, xylenes, and solvents with similar chemical characteristics.
It has been found that the modified and conjugated low weight polymers of the invention may be filtered, separated, and purified with more efficiency and with better results than similarly modified or conjugated high weight polymers of the past. In general, it is easier to separate mixtures of low weight polymers into component species than with similar higher weight polymers.
Endgroup modification and conjugation is generally a multi-step process, with each step of the functionalization resulting in polymeric impurities. If purification is ineffective, which can be the case when dealing with high weight polymers, the impurities accumulate throughout functionalization of the polymer to an unacceptable level. By first performing functionalization and purification processes on a low weight polymer and subsequently joining the purified low weight polymer with a high weight polymer in accordance with this invention, process steps involving the high weight polymer are minimized, resulting in a high weight functionalized or conjugated polymer derivative of overall desirable purity.
Method of Forming the High Molecular Weight Activated Polymer of the Invention
A high molecular weight, water soluble, non-peptidic, polymer having at least one functional group, Z, is covalently attached to a low molecular weight, typically oligomeric, water soluble, non-peptidic, polymer having at least two functional groups, Yxe2x80x2 and Y, which may be the same or different, so long as Y is not reactive with Yxe2x80x2 and Z is reactive with at least one of Y or Yxe2x80x2. The invention also embodies the conjugation of the polymer derivative with a biologically active agent after the functionalized polymer derivative is produced in accordance with the invention.
In general, the method and resulting functionalized polymer are represented by:
(Poly)a-Z+Yxe2x80x2-(Poly)b-Yxe2x86x92(Poly)a-X-(Poly)b-Y
where Polya has at least about 200 repeating units, typically at least about 1000, and more typically between 1000 and 2000; and Polyb has from to about 120, and typically less than 60 repeating units. Y and Yxe2x80x2 can be the same or different, so long as Yxe2x80x2 is not reactive with Y, and are functional groups, and X is a linking molecule formed by the reaction of Z and the Y or Yxe2x80x2 components. In the example above Z is reactive with Yxe2x80x2 to form a linkage X. (Poly)a can be additionally functionalized to produce a homobifunctional polymer with Y reactive groups at each terminus. If the additional group is protected from reaction, the protective group can be removed from the resulting polymer conjugate to create a heterobifunctional polymer.
The resulting polymer derivatives of the invention comprise a high molecular weight water soluble polymer segment which is covalently connected, via a linking group, to a low molecular weight water soluble polymer segment. The low molecular weight polymer segment has a functionalized endgroup, Y, in addition to the endgroup which provides the linkage with the high weight polymer segment. The additional functionalized endgroup, Y, upon the low weight polymer segment, provides a linking group for covalently bonding with a biologically active agent or for conversion to such a group.
The following are offered as non-limiting examples of the invention.